Tumours cannot grow beyond a certain mass without the formation of new blood vessels (angiogenesis), and a correlation between microvessel density and tumour invasiveness has been reported for a number of tumours (Folkman (1995). Nature Med., 1, 27-31). Moreover, angiogenesis underlies the majority of ocular disorders which result in loss of vision [Lee et al., Surv. Ophthalmol. 43, 245-269 (1998); Friedlander, M. et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9764-9769 (1996).]. Molecules capable of selectively targeting markers of angiogenesis would create clinical opportunities for the diagnosis and therapy of tumours and other diseases characterised by vascular proliferation, such as diabetic retinopathy and age-related macular degeneration. Markers of angiogenesis are expressed in the majority of aggressive solid tumours and should be readily accessible to specific binders injected intravenously (Pasqualini et al. (1997). Nature Biotechnol., 15, 542-546; Neri et al. (1997), Nature Biotechnol., 15 1271-1275). Targeted occlusion of the neovasculature may result in tumour infarction and collapse (O'Reilly et al. (1996). Nature Med., 2, 689-692; Huang et al. (1997). Science, 275, 547-550).
The ED-B domain of fibronectin, a sequence of 91 aminoacids identical in mouse, rat and human, which is inserted by alternative splicing into the fibronectin molecule, specifically accumulates around neo-vascular structures (Castellani et al. (1994). Int. J. Cancer 59, 612-618) and could represent a target for molecular intervention. Indeed, we have recently shown with fluorescent techniques that anti-ED-B single-chain Fv antibody fragments (scFv) accumulate selectively in tumoural blood vessels of tumour-bearing mice, and that antibody affinity appears to dictate targeting performance (Neri et al. (1997). Nature Biotechnol., 15 1271-1275; International Patent Application No. PCT/GB97/01412, based on GB96/10967.3). Tumour targeting was evaluated 24 hours after injection, or at later time points.
Various attempts are known in the art to raise antibodies against the ED-B-domain in order to use them for tumour targeting.
Peters et al. (Cell Adhesion and Communication 1995, 3: 67-89) disclose polyclonal antibodies raised to antigens containing no FN sequence other than the intact ED-B domain and show that they bind specifically and directly to this domain.
However, the reagents of Peters et al. suffer from a series of drawbacks:—the antisera of Peters et al. recognise ED-B(+)-FN only after treatment with N-glycanase. This makes these reagents unsuitable for applications such as tumour targeting, imaging and therapy, as deglycosylation cannot be performed in vivo.
The authors acknowledge themselves that their antibodies do not recognise full-length ED-B(+)-FN produced by mammalian cells. They also acknowledge that it had been impossible to produce monoclonal antibodies specific for the ED-B domain of fibronectin, even though antibodies against other domains of fibronectin (such as ED-A) had been produced. It is well-known in the art that polyclonal antisera are unacceptable for above mentioned applications.
Even after years of intense research in this field, monoclonal antibodies recognising the ED-B domain of fibronectin without treatment with N-glycanase could be produced only using phage display techniques as applied in the present invention.
Zang et al. (Matrix Biology 1994, 14: 623-633) disclose a polyclonal antiserum raised against the canine ED-B domain. The authors do expect a cross-reactivity to human ED-B(+)-FN, although this was not tested. However, the authors acknowledge the difficulty to produce monoclonal antibodies directly recognising the ED-B domain of fibronectin (page 631). The antiserum recognises ED-B(+)-FN in Western blot only after treatment with N-glycanase. As mentioned before, glycanase treatment renders these reagents unsuitable for applications according to the present invention.
Recognition of ED-B(+)-FN in ELISA proceeds without the need of deglycosylation but only on cartilage extracted with a denaturing agent (4M Urea) and captured on plastic using gelatin. The authors comment that “the binding of the FN molecule to the gelatine bound on the plastic surface of the ELISA plate may somehow expose the epitopes sufficiently for recognition by the antiserum”. Since for in vivo applications FN cannot be denatured and gelatin bound, the monoclonal binders of the present invention offer distinct advantages.
The Japanese patents JP02076598 and JP04169195 refer to anti-ED-B antibodies. It is not clear from these documents if monoclonal anti ED-B antibodies are described. Moreover, it seems impossible that a single antibody (such as the antibody described in JP02076598) has “an antigen determinant in amino acid sequence of formulae (1), (2) or (3):                (1) EGIPIFEDFVDSSVGY (SEQ ID NO: 22)        (2) YTVTGLEPGIDYDIS (SEQ ID NO: 23)        (3) NGGESAPTTLTQQT (SEQ ID NO: 24)on the basis of the following evidence:        
i) A monoclonal antibody should recognize a well-defined epitope.
ii) The three-dimensional structure of the ED-B domain of fibronectin has been determined by NMR spectroscopy. Segments (1), (2) and (3) lie on opposite faces of the ED-B structure, and cannot be bound simultaneously by one monoclonal antibody.
Furthermore, in order to demonstrate the usefulness of the antibodies localisation in tumours should be demonstrated, as well as evidence of staining of ED-B(+)-FN structures in biological samples without treatment with structure-disrupting reagents.
The BC1 antibody described by Carnemolla et al. 1992, J. Biol. Chem. 267, 24689-24692, recognises an epitope on domain 7 of FN, but not on the ED-B domain, which is cryptic in the presence of the ED-B domain of fibronectin. It is strictly human-specific. Therefore, the BC1 antibody and the antibodies of the present invention show different reactivity. Furthermore, the BC1 antibody recognises domain 7 alone, and domain 7-8 of fibronectin in the absence of the ED-B domain (Carnemolla et al. 1992, J. Biol. Chem. 267, 24689-24692). Such epitopes could be produced in vivo by proteolytic degradation of FN molecules. The advantage of the reagents according to the present invention is that they can localise on FN molecules or fragments only if they contain the ED-B domain.
For the diagnosis of cancer, and more specifically for imaging primary and secondary tumour lesions, immunoscintigraphy is one of the techniques of choice. In this methodology, patients are imaged with a suitable device (e.g., a gamma camera), after having been injected with radiolabelled compound (e.g., a radionuclide linked to a suitable vehicle). For scintigraphic applications, short-lived gamma emitters such as technetium-99m, iodine-123 or indium-111 are typically used, in order to minimise exposure of the patient to ionising radiations.
The most frequently used radionuclide in Nuclear Medicine Departments is technetium-99m (99 mTc), a gamma emitter with half-life of six hours. Patients injected with 99 mTc-based radiopharmaceuticals can typically be imaged up to 12-24 hours after injections; however, accumulation of the nuclide on the lesion of interest at earlier time points is desirable.
Furthermore, if antibodies capable of rapid and selective localisation on newly-formed blood vessels were available, researchers would be stimulated to search for other suitable molecules to conjugate to antibodies, in order to achieve diagnostic and/or therapeutic benefit.